C-reactive protein derived from perivascular adipose tissue accelerates injury-induced neointimal hyperplasia

Figures

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  Fig. 1. Effect of HFD on the expression of CRP in PVAT and confirmation of CRP expression specifically in adipose tissue of CRPTG mice. a mRNA expression of CRP in PVAT was quantified by RT-PCR after WT C57BL6 mice received the HFD for 2 or 8 weeks. b Protein expression of CRP in PVAT was quantified by western blotting after mice were fed the HFD for 8 weeks; the values were normalized to β-tubulin expression as a control. c Gene expression of human CRP was detected by PCR and agarose gel electrophoresis based on genomic DNA extracted from tails of TG mice. d, e Protein expression of human CRP in perivascular adipose tissue (PVAT) was detected by western blotting and immunohistochemical staining. CON control, CRPTG CRP-overexpressing transgenic mice, huCRP human C-reactive protein. Scale bars, 100 μm. *P < 0.05 versus standard diet. Data represent mean ± standard error of the mean (SEM, n = 3)

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  Fig. 2. Overexpression of CRP in PVAT promoted neointimal hyperplasia, adventitial macrophage infiltration, and vasa vasorum proliferation at 4 weeks after endovascular injury. a Representative H&E-stained sections of femoral arteries after endovascular injury in STD-fed mice transplanted with WT PVAT or CRPTG PVAT. Arrows indicate internal elastic lamina. b CD68 (macrophage marker) staining revealed transplanted CRPTG PVAT markedly increased the numbers of adventitial macrophages surrounding injured arteries. Red arrows indicate CD68+ cells. c CD31 (endothelial cell marker, representative of vessels) staining showed transplanted CRPTG PVAT significantly increased adventitial adjacent to the injured vessel. Red arrows indicate CD31+ cells. Scale bars, 100 μm, *P < 0.05 versus WT PVAT transplantation. Data represent mean ± SE (n = 6)

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  Fig. 3. Macrophage infiltration was enhanced in CRPTG adipose tissue in situ and the effect of CRP on the adhesion of macrophage. a Immunohistochemical staining of adipose tissue from WT and CRPTG mice for the macrophage marker CD68 and adipocyte marker TIP47. Representative images and quantitative comparisons of CD68+ cells (n = 6) are shown. Scale bar, 100 μm. b Representative flow cytometric analysis of CD11b + macrophages in vascular stromal fraction isolated from adipose tissue. c Comparison of the expression of inflammatory cytokines between wild-type and CRPTG in perivascular adipose tissue examined by QPCR *P < 0.05 versus wild-type mice. d The effect of recombinant CRP on the adhesion rate of human PMBCs. *P < 0.05 versus 0 μg/mL CRP. e The effect of CRP on the adhesion rate of THP-1 cell-derived macrophages. *P < 0.05 versus BSA control. #P < 0.05 versus 10 μg/mL CRP. Data represent mean ± SEM (n = 6)

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  Fig. 4. Quantitative proteome profiling of adipose tissue from WT and CRPTG mice and bioinformatics analysis of differentially expressed proteins. a Heat map analysis of the WT and CRPTG adipose tissue protein expression profiles, which showed that 137 differentially expressed proteins were detected. b BPGO (Biological Process and Gene Ontology) enrichment analysis of differentially expressed proteins between WT and CRPTG adipose tissues are involved organism processes, response to stimulus, cellular processes, biological regulation and metabolic processes. c Distribution of KEGG pathways. d, e CXCL-7 protein expression detected by quantitative western blot analysis. IB: immunity bolt. GAPDH was used as the loading control. *P < 0.05 versus WT-PVAT. Data represent mean ± SEM (n = 3)

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  Fig. 5. CRP promoted monocyte migration by inducing CXCL7 expression in adipocytes. a After 12 h of starvation, 3T3-L1 adipocytes were stimulated with different concentrations of exogenous CRP recombinant protein. The supernatants were collected 24 h later. CXCL7 production was determined by ELISA and it revealed that CRP dose-dependently increased CXCL7 production in 3T3-L1 adipocytes, with a peak CRP dose of 10 μg/mL. *P < 0.05 versus control. b, c CRP increased CXCL7 protein expression as assessed by western blotting. The values were normalized to β-tubulin expression as a control. *P < 0.05 versus control. d, e Transwell assays with primary human PBMCs plated on the upper cell culture inserts and culture medium alone (control), CRP-treated 3T3-L1 adipocytes plated in the lower chambers in the presence or absence of an anti-CXCL7 antibody at 2 or 4 μg/mL or an isotype-matched IgG control (IgG). Scale bar, 50 μm. *P < 0.05 compared with control (untreated), #P < 0.05 compared with CRP-treated (untreated). Data represent mean ± SEM (n = 6)